title: Mechanism and inhibition of the FabI enoyl-ACP reductase from Burkholderia pseudomallei.
abstract: OBJECTIVE
  As an initial step in developing novel antibacterials against Burkholderia pseudomallei, we have characterized the FabI enoyl-ACP reductase homologues in the type II fatty acid biosynthesis pathway from this organism and performed an initial enzyme inhibition study.
  
  
  METHODS
  A BLAST analysis identified two FabI enoyl-ACP reductase homologues, bpmFabI-1 and bpmFabI-2, in the B. pseudomallei genome, which were cloned, overexpressed in Escherichia coli and purified. Steady-state kinetics was used to determine the reaction mechanism and the sensitivity of bpmFabI-1 to four diphenyl ether FabI inhibitors. The antibacterial activity of the inhibitors was assessed using a wild-type strain of Burkholderia thailandensis (E264) and an efflux pump mutant (Bt38).
  
  
  RESULTS
  Consistent with its annotation as an enoyl-ACP reductase, bpmFabI-1 catalysed the NADH-dependent reduction of 2-trans-dodecenoyl-CoA via a sequential Bi Bi mechanism. In contrast, bpmFabI-2 was inactive with all substrates tested and only bpmfabI-1 was transcriptionally active under the growth conditions employed. The sensitivity of bpmFabI-1 to four diphenyl ethers was evaluated and in each case the compounds were slow-onset inhibitors with K(i) values of 0.5-2 nM. In addition, triclosan and PT01 had MIC values of 30 and 70 mg/L for B. pseudomallei as well as a wild-type strain of B. thailandensis (E264), but MIC values of <1 mg/L for the efflux pump mutant Bt38. A reduction in MIC values was also observed for the pump mutant strain with the other diphenyl ethers.
  
  
  CONCLUSIONS
  Provided that efflux can be circumvented, bpmFabI-1 is a suitable target for drug discovery.
authors.affiliation: Institute for Chemical Biology & Drug Discovery, Department of Chemistry, Stony Brook University, Stony Brook, NY 11794-3400, USA.
authors.forename: Nina
authors.initials: N
authors.lastname: Liu
authors.forename: Jason E
authors.initials: JE
authors.lastname: Cummings
authors.forename: Kathleen
authors.initials: K
authors.lastname: England
authors.forename: Richard A
authors.initials: RA
authors.lastname: Slayden
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Tonge
mesh_terms: D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D016957:Burkholderia pseudomallei / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH) / Q000037:antagonists & inhibitors* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)